Light-Enhanced Hypoxia-Response of Conjugated Polymer

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Biological and Medical Applications of Materials and Interfaces

Light-enhanced hypoxia-response of conjugated polymer nanocarrier for successive synergistic photodynamic and chemo-therapy Xiao-Long Zhang, Ming Wu, Jiong Li, Shanyou Lan, Yongyi Zeng, Xiaolong Liu, and Jingfeng Liu ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b06491 • Publication Date (Web): 08 Jun 2018 Downloaded from http://pubs.acs.org on June 8, 2018

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ACS Applied Materials & Interfaces

Light-enhanced hypoxia-response of conjugated polymer nanocarrier for successive synergistic photodynamic and chemo-therapy Xiaolong Zhanga,d, Ming Wua,d, Jiong Lia,c,d, Shanyou Lana,b, Yongyi Zenga,b,d, Xiaolong Liua,d*, and Jingfeng Liua,b,d*

a. The United Innovation of Mengchao Hepatobiliary Technology Key Laboratory of Fujian Province, Mengchao Hepatobiliary Hospital of Fujian Medical University, Fuzhou 350025, P. R. China b. Liver Disease Center, The First Affiliated Hospital of Fujian Medical University, Fuzhou 350005, P. R. China c. School of Life Sciences, Fujian Agriculture and Forestry University, Fuzhou 350002, P.R. China d. The Liver Center of Fujian Province, Fujian Medical University, Fuzhou 350025, P. R. China KEYWORDS: conjugated camptothecin (CPT)

polymers,

photodynamic

therapy,

chemotherapy,

hypoxia,

ABSTRACT: The tumor hypoxia environment as well as the photodynamic therapy (PDT) induced hypoxia could severely limit the therapeutic efficacy of traditional PDT. Fortunately, the smart integration of hypoxia-responsive drug delivery system with PDT might be a promising strategy to enhance the PDT efficiency that hindered by hypoxia environment. Herein, a novel azobenzene (AZO) containing conjugated polymers (CPs) based nanocarrier (CPs-CPT-Ce6 NPs) was constructed for combination of PDT with chemotherapy, as well as to enhance the hypoxia-responsive drug release by light. The conjugated polymer chains, used as matrix to prepare the CPs-CPT-Ce6 NPs, were benefit for loading hydrophobic photosensitizers and chemotherapy drugs, to improve their cellular uptake. Moreover, the AZO group (-N=N-) in CPs, which can be reduced and cleaved by azoreductase (a typical biomarker of hypoxia) under hypoxia environment of tumor cells, acts as the hypoxia responsive linker component. Upon laser irradiation, the CPs-CPT-Ce6 NPs could produce ROS for PDT then facilitate the enhancement of tumor hypoxic condition, which could further promote the dissociation of CPs via reductive cleavage of AZO bridges to subsequently release cargos (chemotherapeutic drug, CPT), then

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significantly enhance the killing effects to tumor cells that were resistant to PDT. Both in vitro and in vivo studies confirmed the improvement of synergistic therapeutic effects of our CPs-CPT-Ce6 NPs. This cascade responsive approach provides an excellent complementary mode for PDT and could open new insights for constructing programmable and controllable responsive systems in biomedical applications.

1. INTRODUCTION Photodynamic therapy (PDT) has attracted increasing attentions in cancer therapy due to its unique advantages, such as high efficiency, minimal invasion, lack of drug resistance, local light controllable capability and tolerance of repeated doses, when comparing with other conventional therapies.

1–3

Typically, the PDT process involves three key components: photosensitizers (PSs),

molecular oxygen (O2) and light .2 Upon the illumination with light at specific wavelength, PSs can transfer the energy obtained from light to oxygen and then produce cytotoxic reactive oxygen species (ROS) to kill cancer cells.2,3 From this point of view, the highly efficient delivery of PSs to target region is essential for PDT.4 However, most of currently clinical used porphyrin derivate PSs are hydrophobic and tend to aggregate in aqueous solution, which will result in low tumor cell accumulation.5 Therefore, developing effective loading systems to deliver PSs into cancer cells will significantly improve their drug bioavailability and photodynamic therapeutic efficiency. Nanomaterials have shown great prospects for carrying PSs via physical encapsulation, adsorption or chemical conjugation, to improve the cellular uptake of PSs for PDT.6 For example, mesoporous silica nanoparticles,7 gold nanoparticles,8 graphene oxide nanosheets,9 quantum dots10 and manganese dioxide nanoparticles11 have been employed as effective delivery system (DDS) and demonstrated for effective photodynamic cancer therapy. Oxygen is also a crucial factor in PDT treatment. Nevertheless, the continuous intracellular O2 consumption during PDT process will generate acute hypoxia microenvironment and in turn resulting in a considerably limited therapeutic efficacy of PDT.12 To reverse this negative effect and improve the treatment outcomes of PDT, several strategies have been introduced. For instance, delivery of O2 through hyperbaric oxygen or nanomaterials to enhance tumor oxygenation,13,14 production of O2 by decomposing microenvironment H2O2 or O2 self-sufficient nanoplatform to


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relieve hypoxia,15,16 or the combination of other treatment modalities (photothermal therapy or chemotherapy) to further improve the therapeutic efficiency.17–23 In addition, hypoxia is one of the characteristic features within most solid tumors, which is caused by rapid tumor cell proliferation, heterogeneous tumor growth and abnormal tumor vasculature.24,25 It is realized that the development of novel strategies to overcome or utilize the tumor microenvironment (such as pH, RNA, glutathione (GSH), adenosine triphosphate (ATP) or enzymes) for responsive drug delivery holds great promise to improve the therapeutic efficiency of solid cancer treatment.11,20,21,26–28 Notably, the overexpression or activation of intracellular reductases such as azoreductase, nitroreductase and DT-diaphorase can be accelerated by tumor hypoxia, and various hypoxia-responsive motifs based on the reduction of azobenzene group, 2-nitroimidazole group or nitrobenzene group have been designed and used for hypoxic tumor detection or treatment.29–34 Thus, hypoxia microenvironment responsive drug delivery system or prodrugs that can be specifically activated in solid tumors might be one of the effective strategy to overcome hypoxia of tumor. Furthermore, converting the negative effect of hypoxia to a positive factor by combing PDT with hypoxia microenvironment responsive drug-delivery system could be helpful to enhance the PDT therapeutic efficacy. Conjugated polymer nanoparticles (CP NPs) have attracted a great deal of interest in chemistry and biology fields due to their remarkable optical and physical properties.35

For instance, the CP

NPs have been successfully used in applications of cell fluorescence labeling or imaging, in vivo fluorescence or photoacoustic imaging, drug delivery, chemical- or bio-sensors, and cancer therapy.35–38 For PDT or chemotherapy applications, the hydrophobic polymer chains of CP NPs are benefit for loading hydrophobic drugs to enhance their solubility and cellular uptake.39 Moreover, the CP NPs also show excellent biocompatibility due to their features of completely organic and biologically inert.40 Notably, the introducing of responsive linker component to polymer chains of CP NPs will generate specific chemical reactions with the target of interest to further facilitate applications of CP NPs in nanomedicine.41,42 Herein, we report a novel AZO containing conjugated polymer based nanocarrier that was capable of codelivering PSs and chemotherapy drugs for an effective PDT and subsequently hypoxia-responsive drug release for chemotherapy. The responsive nanocarrier was composed of four components: hypoxia-responsive AZO-containing conjugated polymers, photosensitizer



(Ce6), anti-cancer compound (camptothecin, CPT), and polyvinyl pyrrolidone (PVP) based surface coatings. First, the hypoxia-responsive conjugated polymer chain (AZO-CPs) was prepared by reacting the AZO group containing 4,4′-azodianiline with terephthalaldehyde via a condensation reaction (Figure 1A). The AZO group can be reduced and cleaved under hypoxic environment by azoreductase (a typical hypoxia biomarker), which was widely used in imaging agents and bioreductive prodrugs for hypoxic tumors.43,44 Then, the nanocarrier (CPs-CPT-Ce6 NPs) was prepared by a reprecipitation method via mixing the AZO-CPs with CPT and Ce6 in PVP solution (Figure 1B). The CPs NPs can adsorb Ce6 and CPT via hydrophobic or π–π interaction, follow by the PVP coating to enhance the nanocarrier’s dispersibility, leading to improve its applications in biological environment. Upon laser irradiation, the CPs-CPT-Ce6 NPs can convert molecular oxygen to singlet oxygen to kill cancer cells. Then, the rapid consumption of dissolved oxygen by PDT will intensify local hypoxic environment, which could induce the enhancement of expression or activation of reductase in hypoxic tumors.17,31,45 Specifically, the activation of azoreductase under hypoxia condition can lead to the reduction and cleavage of AZO group.31,32,34 Thereafter, the AZO group in CPs could be reduced and cleaved to promote the dissociation of CPs, subsequently release chemotherapeutic drug. The released CPT could inhibit the activity of DNA topoisomerase-I to further kill PDT resistant cancer cells, therefore, our strategy could synergistically enhance the anticancer efficiency of traditional PDT (Figure 1C).

Figure 1. Schematic illustration of the hypoxia-responsive drug-delivery system. (A) Formation of CPs. (B) Formation and the responsive mechanism of CPs-CPT-Ce6 NPs. (C) Schematic


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illustration of the CPs-CPT-Ce6 NPs for ROS generation and hypoxia responsive drug release to enhance the anticancer efficacy.

2. EXPERIMENTAL SECTION 2.1. Materials. 4,4′-azodianiline, terephthalaldehyde, chlorine e6 (Ce6) and camptothecin (CPT) were obtained from J&K Chemical Ltd. Polyvinyl pyrrolidone (PVP, MW.40 k), Na2S2O4 and 2’,7’-dichlorodihydrofluorescein diacetate (DCFH-DA) were purchased from Sigma-Aldrich Chemical Co. Acetic acid (HAc), N, N-Dimethylformamide (DMF) were obtained from Sinopharm Chemical Reagent Co. Ltd (China). Cell Counting Kit (CCK8) was obtained from Dojindo Laboratories (Kumamoto, Japan). Hypoxia/Oxidative Stress Detection Kit was purchased from Enzo Life Sciences Inc. Live-Dead Viability/Cytotoxicity Kit (Calcein AM/EthD-1) and Annexin V-FITC apoptosis detection kit were obtained from Life Technologies, Inc. Cell culture products were purchased from Thermo Fisher Scientific (Gibco, Thermo Fisher Scientific, USA) unless otherwise stated. All other reagents of analytical reagent grade were purchased from Sinopharm Chemical Reagent Co. Ltd and used as received. Ultrapure water purified from a Millipore NanoPure water filtration system (18.2 M·Ω resistivity) was used in all runs. 2.2. Apparatus. Transmission electron microscopy (TEM) images were characterized by a field emission high resolution 2100F TEM (JEOL, Japan). The average size and zeta potential of nanoparticles were measured by DLS using a ZetaSizer (NanoZS, Malvern, UK). Ultraviolet-visible (UV-vis) absorption spectra were collected on a microplate reader (Spectra 206 Max M5, Molecular Devices). Fluorescence spectra were measured on an Agilent Cary Eclipse fluorescence spectrophotometer. Fourier transform infrared (FT-IR) spectra were measured on a Perkin Elmer Spectrophotometer. The confocal fluorescence images were recorded on a confocal fluorescence microscope (Carl Zeiss, LSM 780, Germany). 2.3. Preparation of CPs-CPT-Ce6 NPs. The synthesis of CPs was according to previous reports with

minor

modification,46,47

briefly,

4,4′-azodianiline

(4.2

mg,

0.02

mmol)

and

terephthalaldehyde (2.7 mg ,0.02 mmol) were dissolved in DMF (4 mL) and sonicated for 5 min. Next, aqueous HAc (0.2 mL) was added to the solution under stirring to start the reaction. After 5 min, CPT in DMF (2 mg/mL, 400 µL) was added and further stirred for 10 min. Then, the mixture was drop-wisely added to the PVP aqueous solution (1 mg/mL, 20 mL) under stirring and further



sonicated for 5 min. After that, Ce6 in DMF (2 mg/mL, 400 µL) was added and further stirred for 30 min. The resulting solution of nanoparticles was centrifuged at 6000 rpm for 5 min to remove the bulk particles, and the collected supernatant was further centrifuged at 23000 rpm for 30 min. The obtained precipitates were re-dispersed and washed with ultrapure water by centrifugation at 23000 rpm for several times to remove the unreacted agents. Finally, the resulting CPs-CPT-Ce6 NPs were resuspended in ultrapure water (1 mg/mL) and stored at 4 °C in dark for further usage. The CPs NPs which prepared without the CPT and Ce6, CPs-CPT NPs which prepared without the Ce6, and CPs-Ce6 NPs which prepared without CPT, were prepared at the same way and used as control. The CPs that prepared by reaction of 4,4′-azodianiline and terephthalaldehyde with HAc, followed by adding into water and collected by centrifugation, were characterized by FI-TR or NMR to prove the happening of chemical reactions. The 1H NMR spectra of CPs were recorded on a Varian instrument (400 MHz), and the 1H NMR (400 MHz, DMSO-D6, δ) was including: 8.13 (s, 2H, azobenzene-H), 7.54-7.50 (m, 1H, CH=N), 7.25 (s, 2H, azobenzene-H), 6.71-6.65 (m, 2H, Ar–H). The molecular weight of the CPs was determined by gel permeation chromatography (GPC, Waters, 2414 refractive index detector), with DMF as the eluent and polystyrene as the standard. GPC results: Mn=2160, Mw=5422, polydispersity (PDI)=2.51 (Figure S1). 2.4 Characterization of CPs-CPT-Ce6 NPs. The fluorescence spectra of CPs-CPT-Ce6 NPs were measured for CPT with the excitation wavelength at 360 nm and the emission wavelength ranging from 380 to 550 nm, and for Ce6 with the excitation wavelength at 404 nm and the emission wavelength ranging from 600 nm to 750 nm. The ABDA was used as the ROS indicator to evaluate the ROS generation ability of CPs-CPT-Ce6 NPs by measuring the absorption changes of ABDA as follows: The CPs-CPT-Ce6 NPs (20 µg/mL) were mixed with ABDA (50 µM) in PBS buffer (10 mM, pH 7.4), then the mixture was irradiated by 670 nm laser at the power intensity of 50 mW/cm2 for 0,1,2,5,10 min, and the absorbance of ABDA ranging from 320 nm to 450 nm were recorded. Afterwards, the absorbance of ABDA at 380 nm was used for analysis. The sodium dithionite (2 mM), used as a chemical mimic of azoreductase, mediated azoreductuction of CPs-CPT-Ce6 NPs was performed in PBS (10 mM, pH 7.4) at 37 °C for 60 min. Then the TEM and CPT fluorescence of CPs-CPT-Ce6 NPs before and after reduction were


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recorded. For the stability studies, the CPs-CPT-Ce6 NPs were incubated with H2O, PBS, RPMI-1640 medium and RPMI-1640 medium+10% fetal bovine serum (FBS) for different times, and the size change was measured by DLS. 2.5 Cell culture and fluorescence imaging. HeLa cells and NIH3T3 cells were cultured by RPMI 1640 medium containing 10% FBS and 1% antibiotics of penicillin/streptomycin at 37 °C in a humidified incubator containing 5% CO2. To investigate the cellular uptake of CPs-CPT-Ce6 NPs, HeLa cells were seeded at a density of 1×105 onto 35 mm glass-bottom Petri dish and cultured for 12 h at 37 °C. Thereafter, the nanoparticles were added to the dishes, and further incubated for 4 h. Afterwards, the cells were washed with PBS for three times and then fixed with 4% paraformaldehyde for 15 min at room temperature. Finally, the cells were washed with PBS and imaged by confocal microscope with 404 nm laser excitation for CPT and 633 nm excitation for Ce6. To investigate the intracellular drug release of CPs-CPT-Ce6 NPs under hypoxia or normoxia condition, the cells were first incubated under standard condition (containing humidified 5% CO2/95% air at 37 °C) for 12 h, afterwards the culture plates were transferred into hypoxia (1% O2) or normoxia (5% CO2/95% air) condition for 12 h. The hypoxia condition was made by bubbling the incubator chamber with N2. Thereafter, the nanoparticles were added and further incubated for 4 h under hypoxia or normoxia condition. The following imaging process was the same as above. The laser induced local hypoxic environment and drug release were performed as follows: the cells were incubated under standard condition and then incubated with CPs-CPT-Ce6 NPs for 4 h, afterwards a certain amount of oil (nontoxicity, Enzo Life Sciences) was added on the upper surface of the medium. After 670 nm laser irradiation, the cells were further cultured for 4 h and then imaged by fluorescence microscope. DCFH-DA was used as the ROS fluorescence indicator to evaluate the ROS generation of nanoparticles in cells. After seeding of the HeLa cells on the confocal dish, the corresponding nanoparticles were added and incubated for 4 h. Then, the cells were washed twice with PBS and incubated with DCFH-DA solution (40 µM) for 30 min. Followed by washing with PBS twice, the cells were exposed to 670 nm laser irradiation with the power intensity of 50 mW/cm2 for 10 min. After that, the fluorescence images were observed on a fluorescence microscope with 488 nm excitation.



The therapeutic efficiency of CPs-CPT-Ce6 NPs toward HeLa cells was examined using Live-Dead Viability/Cytotoxicity staining assay. The cells were treated similarly as mentioned above except without addition of the DCFH-DA, then the treated cells were further cultured for 12 h before staining with 2.0 µM Calcein AM for imaging the live cells and 4.0 µM EthD-1 for dead cells under a fluorescence microscope. The nanoparticle (CPs NPs and CPs-Ce6 NPs) mediated intracellular hypoxia generation under light irradiation was measured using Oxidative Stress Detection Kit (Enzo Life Sciences) as follows: The HeLa cells were seeded on a 96-well plate to about 80% confluency. Followed by incubation with nanoparticles (20 µg/mL) for 4 h in the dark, the hypoxia detection mix (according to the manufacturer’s instruction) were added into the cells for 30 min. After washing, HeLa cells were exposed to 670 nm laser with the power intensity of 50 mW/cm2 for 10 min. The untreated group was used as the negative control, and deferoxamine (DFO) treatment was used as the positive control. The hypoxia signal was detected with the excitation wavelength of 561 nm by confocal fluorescence microscope. 2.6 Cytotoxicity assay. To investigate the therapy effect of CPs-CPT-Ce6 NPs, cell counting kit (CCK8) assay was used according to the manufacturer's instructions. Briefly, HeLa cells were seeded on 96 wells plates at 1×104 cells per well for 12 h, and then incubated with 100 µL of nanoparticles (CPs NPs, CPs-CPT NPs, CPs-Ce6 NPs and CPs-CPT-Ce6 NPs) for 4 h in the dark. After washing with PBS twice, one of the nanoparticle treated group was irradiated with 670 nm laser with the power intensity of 50 mW/cm2 for 10 min under normoxia condition. The other groups were cultured without laser irradiation. Afterwards, the cells were further cultured for 24 h before the CCK8 assay. The untreated cells without laser irradiation were used as control. The cytotoxicity assay for different concentration of nanoparticle treatment under hypoxia condition was performed as the same as mentioned above except the cells cultured under hypoxia condition. The therapy effect of CPs-CPT-Ce6 NPs were also examined by Annexin V-FITC/PI Apoptosis Detection Kit. First, the HeLa cells were seeded on 12 wells plates at 1×105 cells per well at 37°C in a 5% CO2 atmosphere for 12h. After washing with PBS twice, HeLa cells were incubated with 500 µL of 20 µg/mL nanoparticles (CPs NPs, CPs-CPT NPs, CPs-Ce6 NPs and CPs-CPT-Ce6 NPs) for 4 h in the dark. After washing with PBS twice, one of the nanoparticle treated group was exposed to 670 nm laser for 10 min, and the other nanoparticle treated groups were cultured under


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dark. Afterwards, the cells were further cultured for 24 h and then stained with Annexin V-FITC/PI in accordance with the manufacturer’s protocol. Finally, the cells were analyzed by flow cytometry (BD FACSverse). The assay for synergistic anticancer effects of CPs-CPT-Ce6 NPs was tested with simulated PDT-induced hypoxia environment according to previous report.17,18 After the cells incubated with nanoparticles and washed by PBS twice, 50 µL of fresh culture medium was added and then 100 µL of oil (nontoxicity, Oxidative Stress Detection Kit, Enzo Life Sciences) was added on the upper surface of the medium. Following the 670 nm laser irradiation at the power intensity of 50 mW/cm2 for 10 min, the cells were further cultured for 24 h and tested by CCK8 assay. 2.7 In vivo anticancer efficacy. All animal experiments were performed in accordance with the guidelines approved by the Animal Ethics Committee of Fujian Medical University. The Male BALB/c-nude mice (six weeks old) were purchased from China Wushi, Inc. (Shanghai, China). The mice were acclimatized one week at the animal facility before the experiment. The HeLa tumor-bearing mice model was established by subcutaneously injecting of a suspension of HeLa cells (1×106) into the right back of the hind leg. When the tumor average diameter reached about 150 mm2, the mice were randomly divided into 5 groups (5 mice per group): (a) control (PBS with laser), (b) CPs NPs with laser irradiation, (c) CPs-Ce6 NPs with laser irradiation, (d) CPs-CPT-Ce6 NPs without laser irradiation and (e) CPs-CPT-Ce6 NPs with laser irradiation. For tumor therapy, 50 µL of PBS or solution of nanoparticles (1 mg/mL) were directly injected into the tumor site at day 0 and 8, respectively. After 4 h, the tumor sites were irradiated with or without 670 nm laser at the power intensity of 150 mW/cm2 for 10 min. The laser irradiation was carried out at the first day and repeated at day 4 and day 8. During the period of treatment, the tumor size change of each mouse was measured every other day. The tumor volume was calculated using the formula, volume = length × (width)2/2. After treatment, the mice were sacrificed, and the tumors were collected and weighted. The tumors from each group of the treated mice were sectioned into slices for histological analysis by H&E and Ki67 staining. For the TUNEL staining, the mice were sacrificed 1 day after therapy, and the tumors were collected, frozen sectioned, and stained according to the manufacturer’s protocols. 2.8 Statistical analysis Quantitative data were shown as the mean ± standard deviation (SD). The statistical analysis of



data between different groups, the one-way of variance (ANOVA) method or the two-tail paired Student’s T-test was performed, *p

Light-Enhanced Hypoxia-Response of Conjugated Polymer

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